Effector Coupling Research Articles

Unraveling allostery within the angiotensin II type 1 receptor for Gαq and β-arrestin coupling.

G protein-coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gαq and β-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gαq coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to β-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred β-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gαq coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gαq and β-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Use of Novel ebBRET Biosensors for Comprehensive Signaling Profiling of One Hundred Therapeutically Relevant Human GPCRs

Functional selectivity is the ability of a given GPCR to engage multiple signaling pathways, with distinct ligands of the given receptor displaying different efficacies in engaging receptor-coupled pathways. Full exploitation of functional selectivity in drug development will require an exhaustive description of the effectors that can be engaged by a given receptor, thus revealing receptor- and ligand-specific signaling signatures. Here, we describe the signaling profiles of 100 therapeutically relevant human GPCRs in response to their endogenous (or prototypical) ligands. Profiling was performed with 15 pathway-selective enhanced bystander bioluminescence resonance energy transfer (ebBRET) biosensors monitoring the activation of specific Gα proteins and βarrestins 1 and 2. The G protein biosensors represent a new generation of BRET-based sensors that measure the translocation of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 dose‑response curves were generated, revealing a great diversity in GPCR coupling selectivity. Our data highlight that the Gi family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Certain GPCRs (17%) showed greater selectivity, with coupling restricted to a single G protein subtype or members of the same G protein family. Others showed broader activation profiles; specifically, 39%, 35% and 9% of GPCRs tested coupled to members of two, three and all four G protein families, respectively. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. Of note, over 50 novel GPCR/G protein couplings were uncovered. In addition to highlighting receptor G protein coupling preferences, our data revealed that 78% of GPCRs recruited βarrestin 1 and/or 2. For certain GPCRs, co‑expression of GRK2 augmented (and even exposed) βarrestin engagement. Finally, in addition to the signaling profiling application, we demonstrated the versatility and usefulness of our ebBRET biosensor platform to study constitutive GPCR activity/inverse agonism, ligand- and SNP-induced biased signaling and cross-talk systems pharmacology. Overall, this work complements and enriches the current body of data on GPCR effector coupling. Moreover, it presents innovative tools allowing to further explore novel GPCR pharmacology. The resources provided in this study, combined to other signaling profiling and omic-scale datasets, will help deconvolute the complexities of GPCR biology and pharmacology and lead to innovative therapeutic exploitation of GPCRs.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Structure-guided design and functional characterization of an artificial red light–regulated guanylate/adenylate cyclase for optogenetic applications

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light–sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light–regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor–effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen–deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor–effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.

Ligand‐specific patterns of PTH1R and arrestin3 internalization and trafficking define a novel form of ligand “bias”

The type 1 parathyroid hormone receptor (PTH1R) couples to Gs, Gq/11, and Gi/o family heterotrimeric G proteins and binds arrestins, which mediate receptor desensitization and arrestin‐dependent signaling. It is known that ligand structure can “bias” PTH1R signaling by changing the efficiency with which it couples to these downstream effectors. Here, we characterize the effect of ligand structure on the signaling, internalization, and intracellular trafficking of human PTH1R in a common HEK293 cell background using a panel of four structurally distinct ligands. Two ligands, hPTH(1‐34) and hPTH(1‐31), behave as conventional PTH1R agonists, activating both Gs‐cAMP and Gq/11‐Ca2+ signaling; while [Trp1]‐PTHrP(1‐36) is a Gs‐cAMP selective agonist; and [D‐Trp12 Tyr34]‐bPTH(7‐34) is a Gs‐cAMP selective inverse agonist. All four promote arrestin‐dependent ERK1/2 activation, with [Trp1]‐PTHrP(1‐36) and [D‐Trp12 Tyr34]‐bPTH(7‐34) exhibiting the greatest degree of arrestin‐dependence. To investigate the effect of ligand structure on PTH1R internalization and trafficking, we employed a “bystander BRET” approach in which the BRET pair consisted of a Renilla luciferase‐tagged Ras‐like small GTPase (Rab) marking a specific intracellular membrane compartment (Rab 1, Rab2, Rab5, Rab7, Rab9 or Rab11) and PTH1R‐YFP or arrestin3‐YFP. Three ligands, hPTH(1‐34), hPTH(1‐31), and [Trp1]‐PTHrP(1‐36) promoted PTH1R‐arrestin3 binding and clathrin‐dependent internalization, while [D‐Trp12 Tyr34]‐bPTH(7‐34), which fails to stabilize a PTH1R‐arrestin3 complex, did not promote internalization. In response to hPTH(1‐34) or hPTH(1‐31), PTH1R transited sequentially through early (Rab5) and late (Rab7/9) endosomes, before distributing between the slow recycling (Rab1/2) trans‐Golgi/ER network and rapid recycling (Rab11) endosome pool. Arrestin3 trafficking paralleled the receptor, consistent with the known role of arrestins in promoting slow PTH1R recycling and degradation. In contrast, PTH1R stimulated with [Trp1]‐PTHrP(1‐36) did not accumulate in Rab5 or Rab7/9 positive endosomes, instead transiting rapidly into the Rab11 and Rab2 positive pools. Notably, arrestin3 did not accompany the PTH1R, indicating that rapid dissociation of the PTH1R‐arrestin complex changed the pattern of PTH1R sorting in favor of receptor recycling. These results suggest that ligand structure, in particular the rate of ligand‐receptor‐arrestin complex dissociation, can “bias” not only the effector coupling profile of a G protein‐coupled receptor, but also it subsequent intracellular trafficking and rate of degradation/resensitization.Support or Funding InformationNIH grants R01 DK055524 (LML), GM095497 (LML); the MUSC FLIPRTETRA Core S10 RR027777 (LML); the Research Service of the Ralph H. Johnson VA Medical Center, Charleston, SC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

G Protein Preassembly Rescues Efficacy of W6.48 Toggle Mutations in Neuropeptide Y2 Receptor

Ligand binding and pathway-specific activation of G protein-coupled receptors is currently being studied with great effort. Individual answers may depend on the nature of the ligands and the effector pathway. Recently, we have presented a detailed model of neuropeptide Y bound to the Y2R. Accordingly, the C-terminal part of the peptide binds deeply in the transmembrane bundle and brings the side chain of the most essential Y36 in close proximity to W6.48 Here, we investigate the role of this interaction for ligand binding and activation of this receptor. BRET sensors were used for detailed investigation of effector coupling and led to the identification of preassembly of the Y2R-Gi complex. It further confirmed ligand-dependent recruitment of arrestin3. Using equally sensitive readouts for Gi activation and arrestin recruitment as well as quantification with operational models of agonism allowed us to identify a strong inherent bias for Gi activation over arrestin3 recruitment for the wild-type receptor. By systematic mutagenesis, we found that W6.48 does not contribute to the binding affinity, but acts as an allosteric connector to couple ligand binding to Gi activation and arrestin3 recruitment. However, even mutagenesis to a small threonine did not lead to a complete loss of signaling. Interestingly, signaling was restored to wild-type levels by ligands that contain a naphthylalanine as the C-terminal residue instead of Y36 Steric and polar contributions of W6.48 for the activation of the receptor are discussed in the context of different mechanisms of G protein coupling and arrestin recruitment.

G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism.

β-Arrestin-Dependent Dopaminergic Regulation of Calcium Channel Activity in the Axon Initial Segment

G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. Here, we find that D3 dopamine receptors (D3R) regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways.

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism

SummaryLigand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Desensitization-resistant and -sensitive GPCR-mediated inhibition of GABA release occurs by Ca2+-dependent and -independent mechanisms at a hypothalamic synapse.

Whereas the activation of Gαi/o-coupled receptors commonly results in postsynaptic responses that show acute desensitization, the presynaptic inhibition of transmitter release caused by many Gαi/o-coupled receptors is maintained during agonist exposure. However, an exception has been noted where GABAB receptor (GABABR)-mediated inhibition of inhibitory postsynaptic currents (IPSCs) recorded in mouse proopiomelanocortin (POMC) neurons exhibit acute desensitization in ∼25% of experiments. To determine whether differential effector coupling confers sensitivity to desensitization, voltage-clamp recordings were made from POMC neurons to compare the mechanism by which μ-opioid receptors (MORs) and GABABRs inhibit transmitter release. Neither MOR- nor GABABR-mediated inhibition of release relied on the activation of presynaptic K(+) channels. Both receptors maintained the ability to inhibit release in the absence of external Ca(2+) or in the presence of ionomycin-induced Ca(2+) influx, indicating that inhibition of release can occur through a Ca(2+)-independent mechanism. Replacing Ca(2+) with Sr(2+) to disrupt G-protein-mediated inhibition of release occurring directly at the release machinery did not alter MOR- or GABAB -mediated inhibition of IPSCs, suggesting that reductions in evoked release can occur through the inhibition of Ca(2+) channels. Additionally, both receptors inhibited evoked IPSCs in the presence of selective blockers of N- or P/Q-type Ca(2+) channels. Altogether, the results show that MORs and GABABRs can inhibit transmitter release through the inhibition of calcium influx and by direct actions at the release machinery. Furthermore, since both the desensitizing and nondesensitizing presynaptic receptors are similarly coupled, differential effector coupling is unlikely responsible for differential desensitization of the inhibition of release.

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.

The G protein‐coupled estrogen receptor (GPER)/GPR30 C‐terminal PDZ motif regulates receptor plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production (662.13)

G protein‐coupled estrogen receptor (GPER), or GPR30, is a G protein‐coupled receptor reported to bind 17β‐estradiol (E2), couple Gαs, and mediate non‐genomic estrogen responses. However, there remains a debate about the receptor pharmacological profile, effector coupling, and membrane trafficking. We addressed receptor membrane trafficking and effects on cAMP production in well‐defined model systems, HEK293 cells and CHO cells, and MDCK cells containing a Cre reporter, transfected to express GPER. We also addressed the role of the receptor C‐terminal type‐I PDZ motif (‐SSAV) by deleting the motif (GPERΔSSAV). E2 and G‐1, a reported GPER agonist, did not stimulate or inhibit cAMP production either in the absence or in the presence of wild‐type (WT) GPER or GPERΔSSAV. However, WT GPER constitutively inhibited forskolin (FSK)‐ and agonist‐stimulated cAMP production in a PDZ‐dependent and pertussis toxin‐insensitive manner. Consistent with this, attenuation of native receptor expression with siRNA increased FSK‐stimulated cAMP production in MDCK cells. Confocal microscopy and cell sorting of HEK293 cells stained with antibody live revealed that both WT GPER and GPERΔSSAV matured to the plasma membrane, but that GPERΔSSAV was subject to much higher constitutive endocytosis. Thus, GPER depends on a PDZ motif for plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production.

Adenosine as a regulator of NFκB activation

Adenosine as a regulator of NF<i>κ</i>B activation

Regulation ofβ2-Adrenergic Receptor Function by Conformationally Selective Single-Domain Intrabodies

The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.

Nicotine modulation of adolescent dopamine receptor signaling and hypothalamic peptide response

Adolescence is a sensitive developmental period for limbic and dopamine systems that coincides with the typical age for onset of tobacco use. We have previously shown that a 4-day, low-dose nicotine (0.06 mg/kg) pretreatment enhances locomotor and penile response to the D2-like agonist, quinpirole (0.4 mg/kg), in adolescent but not adult rats. The present study is designed to determine mechanisms underlying this effect. Nicotine enhancement of adolescent quinpirole-induced locomotion was mediated by D2 receptors (D2Rs) since it was blocked by the D2R antagonist, L-741,626, but not by the D3R and D4R antagonists, NGB 2904 and L-745,870. Enhancement of quinpirole-induced erectile response was blocked by both L-741,626 and NGB 2904, indicating involvement of D3Rs. Whereas D2R binding was unaffected by adolescent nicotine pretreatment, effector coupling in the striatum was increased, as determined by GTPγS binding. Nicotine pretreatment enhanced quinpirole-induced c-fos mRNA expression in the hypothalamic paraventricular and supraoptic nuclei in adolescents only. Adolescent nicotine pretreatment enhanced c-fos mRNA expression in corticotropin releasing factor (CRF) cells of the paraventricular nucleus, and enhancement of penile erection was blocked by the CRF-1 receptor antagonist, CP 376,396. These findings suggest that adolescent dopamine and CRF systems are vulnerable to alteration by nicotine. This is the first evidence for a role of CRF in adolescent erectile response.

Emergent biological properties of arrestin pathway-selective biased agonism

Our growing appreciation of the pluridimensionality of G protein-coupled receptor (GPCR) signaling, combined with the phenomenon of orthosteric ligand “bias”, has created the possibility of drugs that selectively modulate different aspects of GPCR function for therapeutic benefit. When viewed from the short-term perspective, e.g. changes in receptor conformation, effector coupling or second messenger generation, biased ligands appear to activate a subset of the response profile produced by a conventional agonist. Yet when examined in vivo, the limited data available suggest that biased ligand effects can diverge from their conventional counterparts in ways that cannot be predicted from their in vitro efficacy profile. What is currently missing, at least with respect to G protein and arrestin pathway-selective ligands, is a rational framework for relating the in vitro efficacy of a “biased” agonist to its in vivo actions that will enable drug screening programs to identify ligands with the desired biological effects.

Mechanisms of rapid opioid receptor desensitization, resensitization and tolerance in brain neurons

Agonists acting on µ-opioid receptors (MOR) are very effective analgesics but cause tolerance during long-term or repeated exposure. Intensive efforts have been made to find novel opioid agonists that are efficacious analgesics but can elude the signalling events that cause tolerance. µ-Opioid agonists differentially couple to downstream signalling mechanisms. Some agonists, such as enkephalins, D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), methadone and sufentanyl are efficacious at mediating G-protein and effector coupling, as well as triggering MOR regulatory events that include MOR phosphorylation, β-arrestin binding, receptor endocytosis and recycling. By contrast, morphine and closely related alkaloids can mediate efficacious MOR-effector coupling but poorly trigger receptor regulation. Several models have been proposed to relate differential MOR regulation by different opioids with their propensity to cause tolerance. Most are based on dogma that β-arrestin-2 (βarr-2) binding causes MOR desensitization and/or that MOR endocytosis and recycling are required for receptor resensitization. This review will examine some of these notions in light of recent evidence establishing that MOR dephosphorylation and resensitization do not require endocytosis. Recent evidence from opioid-treated animals also suggests that impaired MOR-effector coupling is driven, at least in part, by enhanced desensitization, as well as impaired resensitization that appears to be βarr-2 dependent. Better understanding of how chronic exposure to opioids alters receptor regulatory mechanisms may facilitate the development of effective analgesics that produce limited tolerance.

Time dependence of coupling in frequency-scaled bimanual coordination

Prior research has shown that fluctuations in the relative phase of bimanual coordination do not reflect a white Gaussian noise process. The present study furthered the examination of time-dependent properties in bimanual coordination by comparing the magnitude of relative phase variability and the degree of effector independence within the time domain. The original Kelso (1984) [10] bimanual frequency-scaling protocol was reproduced in which phase transitions from antiphase to in-phase were induced with increasing movement frequency. The results showed that as movement frequency was scaled-up the amount of relative phase variability increased and the effector movements became more dependent prior to the transition. This is consistent with previous modeling showing that stronger effector coupling can prevent the occurrence of phase transitions when long range correlations in relative phase are present. It appears that, as movement frequency is scaled up, increases in effector coupling strength minimize loss of pattern stability and delay the onset of phase transitions.